US20100190210A1 - Protein Purification - Google Patents

Protein Purification Download PDF

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US20100190210A1
US20100190210A1 US12/293,732 US29373207A US2010190210A1 US 20100190210 A1 US20100190210 A1 US 20100190210A1 US 29373207 A US29373207 A US 29373207A US 2010190210 A1 US2010190210 A1 US 2010190210A1
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protein
antibody
interest
exchange chromatography
mixture
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Alahari Arunakumari
Gisela Maria Marques Ferreira
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ER Squibb and Sons LLC
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Medarex LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • This invention relates to protein purification using a two-step non-affinity process comprising two ion exchange steps without the use of an in-process tangential flow filtration step to achieve a high level of purity, which can be formulated for use in human therapy.
  • the invention relates to a method for purifying polypeptides (such as recombinant proteins, antibodies, antibody fragments, Fab and Fv related products, single-chain antibodies, diabodies, linear antibodies, bispecific antibodies, multispecific antibodies including those from antibody fragments, fusion proteins with Fc like regions and antibody-like molecules, e.g. immunoadhesins or peptibodies) from a composition comprising the polypeptide and one or more contaminants.
  • polypeptides such as recombinant proteins, antibodies, antibody fragments, Fab and Fv related products, single-chain antibodies, diabodies, linear antibodies, bispecific antibodies, multispecific antibodies including those from antibody fragments, fusion proteins with Fc like regions and antibody-like molecules, e.g. immuno
  • Therapeutic proteins are typically produced using prokaryotic or eukaryotic cell lines that are engineered to express the protein of interest from a recombinant plasmid containing the gene encoding the protein. Separation of the desired protein from the mixture of components fed to the cells and cellular by-products to an adequate purity, e.g., sufficient for use as a human therapeutic, poses a daunting challenge to biologics manufacturers for several reasons.
  • FDA Food and Drug Administration
  • protein-based pharmaceutical products are substantially free from impurities, including both product related contaminants such as aggregates, fragments and variants of the recombinant protein and process related contaminants such as host cell proteins, media components, viruses, DNA and endotoxins.
  • product related contaminants such as aggregates, fragments and variants of the recombinant protein
  • process related contaminants such as host cell proteins, media components, viruses, DNA and endotoxins.
  • protein purification schemes are available and widely used in the biopharmaceutical industry, they typically include an affinity-purification step, such as Protein A purification in the case of antibodies, in order to reach a pharmaceutically acceptable degree of purity.
  • affinity chromatography is still often employed as a capture step to meet the purity, yield, and throughput requirements for biopharmaceutical antibody purification in order to achieve therapeutic grade purity.
  • affinity chromatography is an expensive purification step for use in purifying therapeutic proteins on a commercial scale.
  • Protein A significantly more expensive than non-affinity media, it also has problems such as resin instability, difficulty with cleaning, ligand leakage, and potential immunogenicity of Protein A or Protein A related compounds contaminating the purified product.
  • affinity media increases the ultimate cost of protein-based therapeutics, particularly those requiring high doses and/or chronic administration.
  • Chromatography alone can account for two thirds of downstream processing costs and, with respect to monoclonal antibodies, the resin cost for an affinity-capture column can overwhelm raw materials cost (see Rathore et al, Costing Issues in the Production of Biopharmaceuticals, BioPharm International, Feb. 1, 2004).
  • HPTFF The purification step of HPTFF, which uses a charged membrane to separate impurities (without limit to relative size), such as proteins, DNA and endotoxins, and to eliminate protein oligomers and degradation products from the mixture containing the antibodies, was essential to achieve the final purity.
  • HPTFF and other traditional TFF steps have disadvantages, namely (1) it is an extra step in the development, optimization and scale-up of the protein therapeutic, which requires membrane cleaning, validation, commercial availability of large-scale GMP cassettes (which is not yet available for HPTFF), and additional buffers, equipment and greater process time, and (2) it increases costs while risking potential loss of product by compromising antibody integrity (by degradation or aggregation or other molecular change that affects molecular activity).
  • non-affinity purification process could remove host cell proteins, nucleic acids, endotoxins, product-related contaminants, e.g., aggregated, oxidized, deamidated or degraded forms of the protein, and media additives, e.g., lipids, vitamins, insulin, methotrexate, amino acids, carbon sources such as glucose, etc.
  • the present invention is based on the unexpected finding that polypeptides and proteins, in particular recombinant proteins such as monoclonal antibodies, can be purified from a contaminated mixture containing contaminants such as host cell proteins and media components by using a two-step purification process that does not include an affinity chromatography step or an in-process buffer exchange step (e.g., TFF or HPTFF). Rather, only pH manipulations and/or dilutions are necessary from the first step to the second step.
  • the two-step process of the present invention therefore, substantially reduces the cost of purifying proteins that typically rely on affinity chromatography or multi-step methodologies to achieve similarly high levels of protein purity.
  • proteins are highly purified to a therapeutic grade using cation exchange chromatography (CEC) and anion exchange chromatography (AEC), without the use of affinity chromatography or an in-process TFF step, in order to yield a high-purity protein composition of greater than 95% monomer that contains negligible amounts of impurities (e.g., host cell proteins, such as CHOPs, in amounts less than 100 parts per million (ppm) and 10 pg/mg or less of nucleic acids).
  • CEC cation exchange chromatography
  • AEC anion exchange chromatography
  • a purification process of the present invention also removes endotoxins, product-related contaminants, such as aggregated, oxidized, deamidated or degraded forms of the protein, and media additives, e.g., lipids, vitamins, insulin, methotrexate, amino acids, carbon sources such as glucose, etc.
  • media additives e.g., lipids, vitamins, insulin, methotrexate, amino acids, carbon sources such as glucose, etc.
  • the present invention provides methods for the purification of recombinant proteins including, but not limited to antibodies, fusion proteins with Fc like regions, and antibody-like molecules, e.g. immunoadhesins, in order to achieve a highly pure protein composition suitable for use in preparing therapeutic grade compositions.
  • recombinant proteins including, but not limited to antibodies, fusion proteins with Fc like regions, and antibody-like molecules, e.g. immunoadhesins
  • the highly pure protein composition is suitable for use in preparing therapeutic grade drug.
  • the invention is a method for purifying a mixture that contains a target protein and one or more contaminants by (a) subjecting the mixture to a CEC purification step followed by an AEC purification step, where there is no in-process TFF step, and (b) isolating the target protein.
  • the AEC step uses an anion exchange membrane.
  • FIG. 1 shows a flow diagram of the two-step purification process of the invention as performed in Example 1.
  • the purification method includes a CEC step followed by a viral inactivation step and an AEC step. Between the CEC and the AEC steps there is a dilution procedure to reduce the conductivity of the CEC elution bulk and obtain the desired viral and contaminant clearance in the AEC step.
  • protein refers generally to peptides and proteins having at least 5 amino acids or more which are linked together by peptide bonds.
  • a protein is typically a complex polypeptide, which can be an antibody, receptor, ligand fusion protein (which comprise at least a portion of two or more polypeptides that are not fused in their natural state), fragments and variants thereof etc., e.g., see US2003022921 and US 20030166869 which are hereby incorporated by reference in their entireties and which list various proteins that can be purified according to a method of the invention.
  • a protein purified according to a method of the present invention can be from any organism (prokaryotic or eukaryotic), particularly from mammals.
  • antibody is used in the broadest sense to include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), immunoadhesins and antibody fragments.
  • An antibody or fragment can be modified such as defucosylated antibody.
  • An antibody can be directed against an “antigen” of interest, such as a polypeptide, which may be a biologically relevant therapeutic target, or a non-polypeptide antigen (e.g, such as tumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178).
  • the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal.
  • Polypeptide antigens include transmembrane molecules (e.g. receptor) and ligands such as growth factors. Exemplary antigens include those polypeptides discussed above.
  • Soluble antigens, or fragments thereof optionally conjugated to other molecules can be used as immunogens for generating antibodies.
  • transmembrane molecules such as receptors
  • fragments of these e.g. the extracellular domain of a receptor
  • cells expressing the transmembrane molecule can be used as the immunogen.
  • Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule.
  • antibody fragment includes at least a portion of a full length antibody and typically an antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; single-chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragments.
  • the term “monoclonal antibody” is used in the conventional sense to refer to an antibody obtained from a population of substantially homogeneous antibodies such that the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. This is in contrast with polyclonal antibody preparations which typically include varied antibodies directed against different determinants (epitopes) of an antigen, whereas monoclonal antibodies are directed against a single determinant on the antigen.
  • the term “monoclonal”, in describing antibodies, indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • monoclonal antibodies used in the present invention can be produced using conventional hybridoma technology first described by Kohler et al., Nature 256:495 (1975), or they can be made using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • Monoclonal antibodies can also be isolated from phage antibody libraries, e.g., using the techniques described in Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991); and U.S. Patent Nos.
  • the monoclonal antibodies described herein include “chimeric” and “humanized” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984)).
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Chimeric or humanized antibodies can be prepared based on the sequence of a murine monoclonal antibody prepared as described above.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g.,. human) immunoglobulin sequences using standard molecular biology techniques.
  • the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.).
  • the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
  • the monoclonal antibodies described herein also include “human” antibodies, which can be isolated from various sources, including, e.g., from the blood of a human patient or recombinantly prepared using transgenic animals.
  • transgenic animals include KM-Mouse® (Medarex, Inc., Princeton, NJ) which has a human heavy chain transgene and a human light chain transchromosome (see WO 02/43478), Xenomouse® (Abgenix, Inc., Fremont Calif.; described in, e.g., U.S. Pat. Nos.
  • Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
  • hypervariable region is used to describe the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (i.e.
  • an immunoadhesin designates antibody-like molecules which combine the “binding domain” of a heterologous “adhesin” protein (e.g. a receptor, ligand or enzyme) with the effector functions of an immunoglobulin constant domain.
  • adhesin e.g. a receptor, ligand or enzyme
  • an immunoadhesin comprises a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is “heterologous”) and an immunoglobulin constant domain sequence.
  • the immunoglobulin constant domain sequence in the immunoadhesin is preferably derived from ⁇ 1, ⁇ 2, or ⁇ 4 heavy chains, since immunoadhesins comprising these regions can be purified by Protein A chromatography (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Immunoadhesins can be purified according to methods of the present invention.
  • a “peptibody” refers to molecules comprising an Fc domain and at least one peptide. Such peptibodies may be multimers or dimers or fragments thereof, and they may be derivatized. Peptibodies are described in greater detail in US Patent Publication No. 20040214190, WO 00/24782 and WO 01/83525, which are incorporated herein by reference in their entireties.
  • ligand binding domain refers to any native cell-surface receptor or any region or derivative thereof retaining at least a qualitative ligand binding of a corresponding native receptor.
  • the receptor is from a cell-surface polypeptide having an extracellular domain which is homologous to a member of the immunoglobulin super gene family.
  • Other receptors which are not members of the immunoglobulin super gene family but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules, e.g. (E-, L- and P-) selectins.
  • receptor binding domain is used to designate any native ligand for a receptor, including cell adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability of a corresponding native ligand. This definition, among others, specifically includes binding sequences from ligands for the above-mentioned receptors.
  • an “antibody-immunoadhesin chimera” comprises a molecule which combines at least one binding domain of an antibody (as herein defined) with at least one immunoadhesin (as defined in this application).
  • Exemplary antibody-immunoadhesin chimeras are the bispecific CD4-IgG chimeras described in Berg et al., PNAS (USA) 88:4723-4727 (1991) and Chamow et al., J. Immunol. 153:4268 (1994).
  • a “mixture” comprises a polypeptide of interest (for which purification is desired) and one or more contaminant, i.e., impurities.
  • the mixture can be obtained directly from a host cell or organism producing the polypeptide.
  • examples of mixtures that can be purified according to a method of the present invention include harvested cell culture fluid, cell culture supernatant and conditioned cell culture supernatant.
  • a mixture that has been “partially purified” has already been subjected to a chromatography step, e.g., non-affinity chromatography, affinity chromatography, etc.
  • a “conditioned mixture” is a mixture, e.g., a cell culture supernatant that has been prepared for a chromatography step used in a method of the invention by subjecting the mixture to one or more of buffer exchange, dilution, salt addition, pH titration or filtration in order to set the pH and/or conductivity range and/or buffer matrix to achieve a desired chromatography performance.
  • a “conditioned mixture” can be used to standardize loading conditions onto the first chromatography column.
  • a mixture can be obtained through various separation means well known in the art, e.g., by physically separating dead and viable cells from other components in the broth at the end of a bioreactor run using filtration or centrifugation, or by concentration and/or diafiltration of the cell culture supernatant into specific ranges of pH, conductivity and buffer species concentration.
  • impurity and contaminant are used interchangeably to mean any material, other than the protein of interest for which it is desirable to have removed from a composition containing the protein of interest.
  • Contaminants include, but are not limited to, any biological macromolecule such as host cell proteins (e.g., CHOPs), polypeptides other than the protein of interest, nucleic acids (e.g., DNA and RNA), lipids, saccharides, endotoxins, bacteria or other microorganisms such as yeast, media components, and any molecule that is part of an adsorbent used in chromatography which may leach into a sample during chromatography, and the like.
  • protein of interest and “target protein” are used interchangeably to refer to a protein, as described above, e.g., an antibody, for which it is desirable to purify from a mixture according to a method of the present invention.
  • host cell protein refers to any of the proteins derived from the metabolism (intra and extra-cellular) of the host cell that expresses the target protein, including any proteins expressed from the genome of the host cell or proteins that are recombinantly expressed, and which are not considered the target protein.
  • the host cell can be any cell that is capable of expressing the target protein, particularly mammalian (e.g., CHO and murine myeloma cell lines such as NSO), insect bacterial, plant and yeast cell lines.
  • the HCP is a “Chinese hamster ovary cell protein”, or “CHOP”, which refers to any of the host cell proteins (“HCP”) derived from a Chinese hamster ovary (“CHO”) cell culture.
  • the HCP is present generally as an impurity in a cell culture medium or lysate [(e.g., a harvested cell culture fluid (“HCCF”)], which contains the protein of interest.
  • HCCF harvested cell culture fluid
  • the amount of HCP present in a mixture comprising a protein of interest provides a measure of the degree of purity for the protein of interest.
  • the amount of HCP in a protein mixture is expressed in parts per million relative to the amount of the protein of interest in the mixture.
  • ppm parts per million
  • purify is used to mean the removal, whether completely or partially, of at least one impurity from a mixture containing the protein and one or more impurities, which thereby improves the level of purity of the protein in the composition (i.e., by decreasing the amount (ppm) of impurity(ies) in the composition).
  • purification is performed with two non-affinity chromatography steps and no in-process TFF steps.
  • a method of the present invention can purify the protein of interest to achieve a composition which contains less than 100 ppm HCP and preferably less than 90 ppm, less than 80 ppm, less than 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm or less than 20 ppm, as determined by ELISA.
  • the term “isolate”, and grammatical variations thereof, refers to the separation of a purified protein of interest from additional substances, e.g., from a column or resin used to purify the protein of interest, in order to achieve a homogenous composition, which contains the protein of interest substantially free from contaminants, impurities and other substances.
  • chromatography refers to the process by which a solute of interest, e.g., a protein of interest, in a mixture is separated from other solutes in the mixture by percolation of the mixture through an adsorbent, which adsorbs or retains a solute more or less strongly due to properties of the solute, such as pI, hydrophobicity, size and structure, under particular buffering conditions of the process.
  • a solute of interest e.g., a protein of interest
  • an “adsorbent” is any solid and fixed substance capable of adsorbing another substance to its surface by adhesion by either direct interaction with the molecule of interest or interaction with compounds that are attached to the adsorbent.
  • Adsorbents that are useful in various types of chromatography are well known in the art and are readily available through commercial sources.
  • affinity chromatography and “protein affinity chromatography” are used interchangeably to refer to a protein separation technique in which a protein of interest is reversibly and specifically bound to a biospecific ligand, usually as a combination of spatial complementarity and one or more types of chemical interactions, e.g., electrostatic forces, hydrogen bonding, hydrophobic forces, and/or van der Waals forces at the binding site. These interactions are not due to the general properties of the molecule such as isoelectric point, hydrophobicity or size but are a result of specific interactions from the molecule of interest and the ligand such as the hydrophobic and precise protein domain fit for protein A and antibody interactions, for example.
  • Protein A is an example of an adsorbent, which can be fixed to a solid support, e.g., Sepharose, for binding molecules that contain an F C region. See Ostrove (1990) in Guide to Protein Purification, Methods of Enzymology 182: 357-379, which is incorporated herein by reference in its entirety.
  • any ligand can be used to purify its respective specific binding protein.
  • the biospecific ligand is covalently attached to a chromatographic solid phase material and is accessible to the protein of interest (e.g. antibody, enzyme, or receptor protein) in solution as the solution contacts the chromatographic solid phase material.
  • the protein of interest retains its specific binding affinity for the biospecific ligand (antigen, substrate, cofactor, or hormone, for example) during the chromatographic steps, while other solutes and/or proteins in the mixture do not bind appreciably or specifically to the ligand.
  • Binding of the protein of interest to the immobilized ligand allows contaminating proteins and other impurities to be passed through the chromatographic medium while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material.
  • the specifically bound protein is then removed form the immobilized ligand with low pH, high pH, low salt, high salt, competing ligand, or the like, and passes through the chromatographic column with the elution buffer.
  • Contaminating proteins having a lower relative concentration to the protein of interest, and other types of contaminants such as nucleic acids and endotoxin, that were earlier allowed to pass through the column may also be present.
  • binding and binding specificity describe the generally specific and reversible interactions between a protein of interest and a ligand requiring the combined effects of spatial complementarity of protein and ligand structures at a binding site coupled with one or more type of electrostatic forces, hydrogen bonding, hydrophobic forces, and/or van der Waals forces at the binding site.
  • the ligand should have chemically modifiable groups which allow it to be attached to the matrix without destroying its binding activity.
  • the ligand should ideally have an affinity for the binding substance in the range 10 ⁇ 4 to 10 ⁇ 8 M in free solution.
  • Non-limiting examples of specific binding include antibody-antigen binding, enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation, DNA binding protein-DNA binding, regulatory protein-protein interactions, and the like.
  • non-affinity chromatography and “non-affinity purification” refer to a purification step which does not use affinity chromatography, but rather requires a non-specific binding interaction between a solute (e.g., protein of interest) and the adsorbent matrix.
  • solute e.g., protein of interest
  • non-specific binding refers to the interactions between a protein of interest and a ligand or other compound bound to a solid phase matrix through non-specific interactions, e.g., through electrostatic forces, hydrogen bonding, hydrophobic forces, and/or van der Waals forces at an interaction site, but lacking the structural complementarity that enhances the effects of the non-structural forces such as in affinity (specific) binding.
  • chromatography processes that rely on non-specific binding, rather than affinity, include ionic exchange chromatography (e.g., anionic and cationic exchange) and hydrophobic charge induction chromatography.
  • hydrophobic charge induction chromatography is a type of mixed mode chromatographic process in which the protein of interest in the mixture binds to a dual mode (i.e., there is one mode for binding and another mode for elution), ionizable ligand [see Boschetti et al., 2000, Genetic Engineering News 20(13)] through mild hydrophobic interactions in the absence of added salts (e.g. a lyotropic salts).
  • a “hydrophobic charge induction chromatography resin” is a solid phase that contains a ligand which has the combined properties of thiophilic effect (i.e., utilizing the properties of thiophilic chromatography), hydrophobicity and an ionizable group for its separation capability.
  • an HCIC resin used in a method of the invention contains a ligand that is ionizable and mildly hydrophobic at neutral (physiological) or slightly acidic pH, e.g., about pH 5 to 10, preferably about pH 6 to 9.5. At this pH range, the ligand is predominantly uncharged and binds a protein of interest via mild non-specific hydrophobic interaction. As pH is reduced, the ligand acquires charge and hydrophobic binding is disrupted by electrostatic charge repulsion towards the solute due to the pH shift.
  • Suitable ligands for use in HCIC include any ionizable aromatic or heterocyclic structure and dyes, including derivatives thereof; see Burton and Harding, Journal of Chromatography A 814: 81-81 (1998) and Boschetti, Journal of Biochemical and Biophysical Methods 49: 361-389 (2001), which includes an aliphatic chain and at least one sulfur atom on the linker arm and/or ligand structure.
  • An example of an HCIC resin includes MEP HYPERCEL (Pall Corporation; East Hills, N.Y.).
  • ion-exchange and ion-exchange chromatography refer to a chromatographic process in which an ionizable solute of interest (e.g., a protein of interest in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate conditions of pH and conductivity, such that the solute of interest interacts non-specifically with the charged compound more or less than the solute impurities or contaminants in the mixture.
  • the contaminating solutes in the mixture can be washed from a column of the ion exchange material or are bound to or excluded from the resin, faster or slower than the solute of interest.
  • Ion-exchange chromatography specifically includes cation exchange, anion exchange, and mixed mode chromatographies.
  • ion exchange material refers to a solid phase that is negatively charged (i.e. a cation exchange resin) or positively charged (i.e. an anion exchange resin).
  • the charge can be provided by attaching one or more charged ligands (or adsorbents) to the solid phase, e.g. by covalent linking.
  • the charge can be an inherent property of the solid phase (e.g. as is the case for silica, which has an overall negative charge).
  • a “cation exchange resin” refers to a solid phase which is negatively charged, and which has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Any negatively charged ligand attached to the solid phase suitable to form the cation exchange resin can be used, e.g., a carboxylate, sulfonate and others as described below.
  • cation exchange resins include, but are not limited to, for example, those having a sulfonate based group (e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast FlowTM, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, Macro-Prep High S from BioRad, Ceramic HyperD S, Trisacryl M and LS SP and Spherodex LS SP from Pall Technologies,); a sulfoethyl based group (e.g., Fractogel SE from EMD, Poros S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, Poros HS-20 and HS 50 from Applied Biosystems); a sulfoisobutyl based group (e.g., Fractogel
  • a carboxylic acid based group e.g., WP CBX from J. T Baker, DOWEX MAC-3 from Dow Liquid Separations, Amberlite Weak Cation Exchangers, DOWEX Weak Cation Exchanger, and Diaion Weak Cation Exchangers from Sigma-Aldrich and Fractogel EMD COO- from EMD
  • a sulfonic acid based group e. g., Hydrocell SP from Biochrom Labs Inc., DOWEX Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP Sulfonic from J. T.
  • a cation exchange membrane can be used instead of a cation exchange resin, e.g., Sartobind S (Sartorius; Edgewood, N.Y.).
  • anion exchange resin refers to a solid phase which is positively charged, thus having one or more positively charged ligands attached thereto.
  • Any positively charged ligand attached to a solid phase suitable to form the anionic exchange resin can be used, such as quaternary amino groups.
  • a ligand used in AEC can be a quaternary ammonium, such as quaternary alkylamine and quaternary alkylalkanolamine, or amine, diethylamine, diethylaminopropyl, amino, timethylammoniumethyl, trimethylbenzyl ammonium, dimethylethanolbenzyl ammonium, and polyamine.
  • a membrane having a positively charged ligand such as a ligand described above, can be used instead of an anion exchange resin.
  • anion exchange resins include, but are not limited to, DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEXTM and FAST Q SEPHAROSETM from GE Healthcare, WP PEI, WP DEAM, WP QUAT from J. T.
  • DMAE DMAE from EMD
  • Amberlite weak and strong anion exchangers type I and II DOWEX weak and strong anion exchangers type I and II
  • Diaion weak and strong anion exchangers type I and II Duolite from Sigma-Aldrich
  • TSK gel Q and DEAE 5PW and 5PW-HR TSK gel Q and DEAE 5PW and 5PW-HR
  • Toyopearl SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C from Tosoh and QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D and Express-Ion Q from Whatman.
  • anion exchange membrane can be used instead of an anion exchange resin.
  • anion exchange membranes include, but are not limited to, Sartobind Q from Sartorius, Mustang Q from Pall Technologies and Intercept Q membrane from Millipore.
  • a “mixed mode ion exchange resin” refers to a solid phase which is covalently modified with cationic, anionic, and/or hydrophobic moieties.
  • Examples of mixed mode ion exchange resins include BAKERBOND ABXTM (J. T. Baker, Phillipsburg, N.J.), ceramic hydroxyapatite type I and II and fluoride hydroxyapatite (BioRad; Hercules, Calif.), Capto MMC (GE Healthcare; Waukesha, Wis.) and MEP and MBI HyperCel (Pall Corporation; East Hills, N.Y.).
  • thiophilic refers to the selectivity that proteins have for sulfone groups that lie in close proximity to thioether groups (Porath et al, 1985).
  • Thiophilic chromatography also known as “thiophilic adsorption chromatography”, is a type of non-affinity chromatography in which a protein of interest, which contains thiophilic regions and aromatic amino residues, bind to a sulphur containing ligand for the isolation of the protein.
  • a thiophilic gel can be prepared by reducing divinylsulfone(coupled to Sepharose 4B) with ⁇ -mercaptoethanol.
  • Thiophilic adsorption chromatography is based on electron donor-acceptor properties and is distinct from chromatography based on hydrophobicity. Hydrophobic associations and ionic interactions do not occur with thiophilic sorbents since thio-ethylsulfone structures do not possess pronounced hydrophobicity or ionic charges. Examples of commercially available thiophilic chromatography resins include Fractogel EMD TA (Merck KGaA; Darmstadt, Germany), Uniflow and Superflow resin (Clontech; Mountain View, Calif.) and T-Gel (Pierce; Rockford, Ill.).
  • solid phase is used to mean any non-aqueous matrix to which one or more ligands. can adhere or alternatively, in the case of size exclusion chromatography, it can refer to the gel structure of a resin.
  • the solid phase can be any matrix capable of adhering ligands in this manner, e.g., a purification column, a discontinuous phase of discrete particles, a membrane, filter, gel, etc.
  • materials that can be used to form the solid phase include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (e.g. controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of these.
  • the present invention is not limited to any particular solid phase material for use in a chromatography step, and those having ordinary skill in the art will be able to select appropriate solid phase material for use in the present invention.
  • Membrane as used herein to refer to a type of chromatography, is generally a polymeric microfilter, such as cellulose acetate and polyvinylidene difluoride, having absorption properties. It is an advantage to use a membrane instead of resin in chromatography since there is no need for packing, qualifying, validating or running cleaning and recycle studies. Moreover, a membrane offers much higher clearance capacity values, e.g., higher than 200-fold compared with its resin counterpart, and high flow rate capability resulting in cycles of only minutes, which significantly shortens the time needed for contaminant removal.
  • Membranes can be used in fast chromatography using step elution or as a flowthrough mode in which very large volumes can be processed. As a result, buffer usage is minimized by up to 90%, compared with resin, resulting in a significant cost-savings.
  • membrane chromatography technology offers process scale removal of DNA at flow rates as high as 100 times faster than traditional chromatography methods, enabling processing of large volumes per manufacturing shift.
  • detergent refers to ionic, zwitterionic and nonionic surfactants, which are useful for preventing aggregation of proteins and to prevent non-specific interaction or binding of contaminants to the protein of interest, and can be present in various buffers used in the present invention, including sanitization, equilibration, loading, post-load wash(es), elution or strip buffers.
  • a detergent is added to a wash buffer.
  • detergents that can be used in the invention include, but are not limited to polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g.
  • poloxamer 188 Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.
  • lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; MONAQUAT series (Mona Industries, Inc., Paterson, N.J.); Igepal CA-630, Pluronic, Triton, BRIJ, Atlas G2127, Genapol, HECAMEG, LUBROL PX, MEGA, NP, THESIT, TOPPS, CHAPS, CHAPSO, DDMAU, EMPIGEN BB, AWITTERGENT and C12E8.
  • MONAQUAT series Mona Industries, Inc., Paterson, N.J.
  • Igepal CA-630 Pluronic, Triton, BRIJ, Atlas G2127, Genapol, HECAMEG, LUBROL PX, MEGA, NP, THESIT, TOPPS, CHAPS, CHAP
  • the detergent can be added in any working buffer and can also be included in the feed containing the molecule of interest.
  • Detergents can be present in any amount suitable for use in a protein purification process, e.g., from about 0.001% to about 20% and typically from about 0.01% to about 1%.
  • polysorbate 80 is used in a wash buffer for CEC.
  • a “buffer” used in the present invention is a solution that resists changes in pH by the addition of acid or base by the action of its acid-base conjugates components.
  • Various buffers can be employed in a method of the present invention depending on the desired pH of the buffer and the particular step in the purification process [see Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975)].
  • Non-limiting examples of buffer components that can be used to control the pH range desirable for a method of the invention include acetate, citrate, histidine, phosphate, ammonium buffers such as ammonium acetate, succinate, MES, CHAPS, MOPS, MOPSO, HEPES, Tris, and the like, as well as combinations of these TRIS-malic acid-NaOH, maleate, chloroacetate, formate, benzoate, propionate, pyridine, piperazine, ADA, PIPES, ACES, BES, TES, tricine, bicine, TAPS, ethanolamine, CHES, CAPS, methylamine, piperidine, o-boric acid, carbonic acid, lactic acid, butaneandioic acid, diethylmalonic acid, glycylglycine, HEPPS, HEPPSO, imidazole, phenol, POPSO, succinate, TAPS, amine-based, benzylamine, trimethyl or di
  • the pH and conductivity of the buffers can vary depending on which step in the purification process the buffer is used. Any suitable buffer at a pH compatible with the selected ligand and resin/membrane can be used for purifying the protein of interest, such as the buffers described above.
  • the pH of the buffer can be between 3 and 10, more preferably from about pH 4.0 to 9.0; conductivity can be from about 0.1 to 40 mS/cm, more preferably from about conductivity 0.5 to 15mS/cm, depending on the purification step and the buffer employed.
  • the pH of the buffers can be from about 4 to 10, more preferably from about pH 6.0 to 9.0; conductivity can be from about 0.1 to 10.0 mS/cm, more preferably from about 0.5 to 5 mS/cm, depending on the purification step and the buffer employed. Buffers used throughout the purification process are described in greater detail below for each chromatography step.
  • a “sanitization” solution is typically used to clean the resin used in column chromatography by removing any bound contaminants, e.g., those of biological origin, prior to the purification process.
  • Any desirable buffer could be used for this purpose provided it is compatible with the particular column and resin selected according to a method of the invention.
  • the pH of the sanitization solution is high, e.g., pH 10 or greater, more preferably pH 11 or greater, and still more preferably pH 12 or greater, alternatively, the pH of the sanitization solution can be low, e.g. pH 4 or less, more preferably pH 3 or less.
  • a resin used in a method of the invention is cleaned using a sanitization solution that includes IN NaOH, pH ⁇ 12.
  • An “equilibration buffer” is used to adjust the pH and conductivity of the chromatography medium, e.g., a resin or membrane, used in CEC or AEC prior to loading the resin with the mixture containing the protein of interest for purification.
  • Suitable buffers that can be used for this purpose are well known in the art, e.g., such as buffers described above, and include any buffer at pH that is compatible with the selected resin used in the chromatography step for purifying the protein of interest.
  • the equilibration buffer species for CEC and AEC are phosphate or TRIS based.
  • the equilibration buffer has a conductivity and/or pH such that the polypeptide of interest is bound to the resin or such that the protein of interest flows through the column while one or more impurities bind to the column, depending on whether CEC or AEC is used.
  • equilibration is completed when the pH and conductivity of the chromatography medium are within ⁇ 0.2 and ⁇ 0.4 mS/cm of the equilibrating buffer, respectively, more preferably within ⁇ 0.1 and ⁇ 0.2 mS/cm of the equilibrating buffer, respectively.
  • the pH of the equilibration buffer is from about 3 to about 9, more preferably pH from about 4.0 to about 8.0, conductivity from about 0.1 to about 40 mS/cm, more preferably from about 0.5 to about 10.0 mS/cm.
  • the pH of the equilibration buffer is from about 4 to about 10, more preferably pH from about 6 to 9, conductivity from about 0.1 (WFI) to about 10 mS/cm, more preferably conductivity from about 0.5 to about 5 mS/cm.
  • a “loading buffer” is used to load the mixture containing the protein of interest onto the column. It shall be appreciated that if a membrane is used as the chromatography medium then the loading buffer is simply contacted with the membrane according to conventional methods used in the art. Any appropriate buffered solution can be used as the loading buffer.
  • the loading buffer is a phosphate or TRIS buffer.
  • the conductivity and pH of the loading buffer is selected such that the protein of interest is bound to the chromatography medium while contaminants are able to flow through.
  • the conductivity and pH of the loading buffer is selected such that the protein of interest is able to flow through while contaminants are retained by the chromatography medium.
  • Suitable buffers for use as a loading buffer are well known in the art, e.g., such as those described above. It shall be appreciated by those having ordinary skill in the art that loading buffers for CEC and AEC can be used at comparable (if not the same) pH and conductivities as described above for the equilibration buffers for CEC and AEC.
  • wash buffer or “post load wash”, as used herein, refer to a buffer used to remove impurities from a chromatography resin (e.g., when using a column) prior to eluting the protein of interest.
  • the term “washing”, and grammatical variations thereof, is used to describe the passing of an appropriate wash buffer through or over the chromatography resin.
  • a wash buffer is used in CEC and can be used in AEC to “push” the product out the column but is not required.
  • a wash buffer is unnecessary in membrane AEC since the protein of interest is not retained by the AEC membrane medium. If desirable, the wash, equilibration and loading buffers can be the same.
  • the pH and conductivity of the wash buffer used in CEC is such that one or more impurities are eluted from the resin while the resin retains the polypeptide of interest.
  • the wash buffer can contain a detergent, as described above, such as a polysorbate. It is important to select pH and conductivity of the wash buffer to remove HCPs and other contaminants without significantly eluting the protein of interest.
  • the pH and conductivity conditions described above for equilibration and loading buffers are sufficient to achieve this goal.
  • the conductivity and pH can be reduced, or maintained or increased in wash buffers used in subsequent wash steps for CEC after loading the mixture in order to remove more hydrophilic and more acidic or basic contaminants than that of the protein of interest and to reduce the conductivity of the system prior to the elution step.
  • the pH and conductivity of the wash buffer are selected such that the protein of interest is retained in the CEC resin used in the process.
  • buffers suitable for use as a wash buffer are described above.
  • the wash buffer is phosphate or TRIS based.
  • the pH of the wash buffer used in CEC can be from about 3 to about 10, more preferably pH from about 4 to about 9; and conductivity from about 0.1 to about 40 mS/cm, more preferably conductivity from about 0.5 to about 5 mS/cm.
  • elute buffer refers to a buffer used to elute the protein of interest from the CEC resin.
  • elute and grammatical variations thereof, refers to the removal of a molecule, e.g., polypeptide of interest, from a chromatography material by using appropriate conditions, e.g., altering the ionic strength or pH of the buffer surrounding the chromatography material, by addition of a competitive molecule for the ligand, by altering the hydrophobicity of the molecule or by changing a chemical property of the ligand (e.g. charge), such that the protein of interest is unable to bind the resin and is therefore eluted from the chromatography column.
  • appropriate conditions e.g., altering the ionic strength or pH of the buffer surrounding the chromatography material, by addition of a competitive molecule for the ligand, by altering the hydrophobicity of the molecule or by changing a chemical property of the ligand (e.g. charge), such that the protein of interest is unable
  • the term “eluate” refers to the effluent off the column containing the polypeptide of interest when the elution is applied onto the column. After elution of the polypeptide of interest the column can be regenerated, sanitized and stored as needed.
  • the pH and conductivity of the elution buffer are selected such that the protein of interest is eluted from the CEC resin used in the process.
  • Examples of buffers suitable for use as an elution buffer are described above.
  • the elution buffer is phosphate or TRIS based.
  • the pH of the elution buffer used in CEC can be from about 3 to about 10, more preferably pH from about 4 to about 9; and conductivity from about 0.1 to about 40 mS/cm, more preferably conductivity from about 5 to about 15 mS/cm.
  • a “regeneration solution” can be used to “strip” or remove tightly bound contaminants from a column used in the purification process.
  • the regeneration solution has a conductivity and pH sufficient to remove substantially any remaining impurities and protein of interest from the resin.
  • the pH of the equilibration buffer used in membrane AEC can be from about 4 to about 10, more preferably pH from about 6 to about 9; and conductivity from about 0.1 to about 10 mS/cm, more preferably conductivity from about 0.5 to about 5 mS/cm.
  • the term “conductivity” refers to the ability of an aqueous solution to conduct an electric current between two electrodes at a particular temperature. A current flows by ion transport in solution. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity.
  • the temperature at which purification is typically performed can be from about 4 to about 37° C., more preferably from about 15 to about 25° C. within the specified pH ranges.
  • the unit of measurement for conductivity is milliSiemens per centimeter (mS/cm), and can be measured using a standard conductivity meter.
  • the conductivity of a solution can be altered by changing the concentration of ions therein.
  • concentration of a buffering agent and/or concentration of a salt (e.g. NaCl or KCl) in the solution may be altered in order to achieve the desired conductivity.
  • the salt concentration is modified to achieve the desired conductivity as described in the Example below.
  • the “pI” or “isoelectric point” of a polypeptide refers to the pH at which the polypeptide's positive charge balances its negative charge.
  • the pI can be calculated according to various conventional methodologies, e.g., from the net charge of the amino acid and/or sialic acid residues on the polypeptide or by using isoelectric focusing.
  • a “low pH hold” refers to a decrease in the pH of an eluate which contains the protein of interest, where the pH decrease is to less than about pH 5, preferably less than about pH 4, more preferably less than about pH 3.7, and most preferably about pH 3.4 to about 3.6 in order to achieve viral inactivation (i.e., at least a 2 log reduction in viral titer, more preferably more than 3 log reduction) followed by an increase in pH in order to prepare the eluate for the second chromatography step or to bring the product to a matrix that provides stability to the molecular integrity of the product of interest.
  • viral inactivation i.e., at least a 2 log reduction in viral titer, more preferably more than 3 log reduction
  • Any suitable acid can be applied to the eluate containing the protein of interest to decrease the pH for the low pH hold, e.g., 1 N HCl or glacial acetic acid based solution.
  • Any suitable base can be applied to the eluate to return its pH to more neutral ranges, e.g., IN NaOH or IN Tris.
  • TFF Tangential flow filtration
  • crossflow filtration refers to a filtration process in which the sample mixture circulates across the top of a membrane, while applied pressure causes certain solutes and small molecules to pass through the membrane.
  • the solution flows parallel to the filter membrane.
  • a pressure differential across the membrane causes fluid and filterable solutes (whose molecular weight is smaller than that of the membranes or behaves like so, such as globular proteins) to flow through the filter. This can be conducted as a continuous-flow process, since the solution is passed repeatedly over the membrane while the fluid that passes through the filter is continually drawn off into a separate circuit.
  • TFF high performance tangential flow filtration
  • the membrane is charged, therefore using both size and charge of molecules to separate contaminants (see US Patent Pub. No. 2003/0229212).
  • TFF can be used to exchange the buffer in which the protein of interest is solubilized into another buffer that is more suitable for binding onto the chromatography resin prior to beginning a method of the present invention.
  • an in-process TFF step i.e., TFF occurs between the two chromatography steps
  • TFF occurs between the two chromatography steps
  • in-process refers to any methodology, step, operation, etc. that is performed after commencing the capture step using CEC and prior to completion of collection of the protein of interest during AEC.
  • An in-process methodology can directly result in a change in purity (e.g., an in-process TFF), but is not required to result in a change in purity (e.g., pH and/or conductivity manipulations).
  • a therapeutic grade of protein purity may be desirable to achieve a therapeutic grade of protein purity using a method of the invention, in which case additional steps well-known in the art can be optionally employed.
  • additional steps well-known in the art can be optionally employed.
  • low pH inactivation and viral filtration methods including charged membrane filtration (e.g., VR CUNO from CUNO, DV20 from Pall Technologies and Planova filters from Asahi), can be used.
  • depth filtration is a filtration method thatuses depth filters, which are typically characterized by their design to retain particles within a filter matrix.
  • the depth filter's capacity is typically defined by the depth, e.g. 10 inch or 20 inch of the matrix and thus the holding capacity for solids.
  • a depth filter can be used to improve the viral clearance capacity of the purification scheme, however it is an optional step and not required to achieve purity levels of a method of the present invention.
  • the depth filter for virus removal can be used at any point during the purification scheme, but is preferably used after the first chromatography step with low process volumes, due to the cost of the filter.
  • CEC followed by AEC are used to purify a protein of interest to achieve a purity of as little as about 100 ppm HCP. or less, 10 pg/mg DNA or less, and as high as 99% monomer purity or greater. Neither TFF nor additional chromatography steps are needed to obtain this high level of purity.
  • the protein of interest can be produced or expressed by living host cells that have been genetically engineered to produce the protein.
  • Methods of genetically engineering cells to produce proteins are well known in the art. See e.g. Ausabel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York) and U.S. Pat. Nos. 5,534,615 and 4,816,567, each of which is specifically incorporated herein by reference.
  • Such methods include introducing nucleic acids that encode and allow expression of the protein into living host cells.
  • These host cells can be bacterial cells, fungal cells, or, preferably, animal cells grown in culture.
  • Bacterial host cells include, but are not limited to E. coli cells. Examples of suitable E.
  • coli strains include: HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleave foreign DNA.
  • Fungal host cells that can be.used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells.
  • a few examples of animal cell lines that can be used are CHO, VERO, DXB11, BHK, HeLa, Cos, MDCK, 293, 3T3, NSO and W1138. New animal cell lines can be established using methods well know by those skilled in the art (e.g., by transformation, viral infection, and/or selection).
  • the protein of interest is produced in a CHO cell (see, e.g., WO 94/11026).
  • CHO cells Various types of CHO cells are known in the art, e.g., CHO-K1, CHO-DG44, CHO-DXB11, CHO/dhfr and CHO-S.
  • a host cell that has been engineered with nucleic acid encoding the protein of interest can be cultured under conditions well known in the art that allow expression of the protein.
  • Preparation of a mixture for protein purification from cellular debris initially depends on the manner of expression of the protein. Some proteins are caused to be secreted directly from the cell into the surrounding growth media, while other proteins are retained intracellularly. For such proteins produced intracellularly, the cell can be disrupted using any of a variety of methods, such as mechanical shear, osmotic shock, and enzymatic treatment. The disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments which can be removed by centrifugation or by filtration. A similar problem arises, although to a lesser extent, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins during the course of the protein production run.
  • the protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.
  • a method of the invention does not rely on any particular methodology to remove cellular debris. Any method can be employed by the skilled practitioner to accomplish this.
  • the particulate debris either host cells or lysed fragments, can be removed, for example, by a centrifugation or filtration step in order to prepare a mixture for purification.
  • the recombinant host cells may be separated from the cell culture medium by, e.g., tangential flow filtration (TFF) or depth filtration, in order to prepare a mixture for purification.
  • TMF tangential flow filtration
  • a mixture containing the protein of interest i.e., the protein has been recovered after expression under appropriate host cell culturing conditions
  • its separation from contaminants in the mixture is performed using a combination of CEC and AEC (see FIG. 1 ), under appropriate conditions, as described hereinabove.
  • No other in-process purification steps, e.g., TFF or HPTFF, are required to achieve levels of purity attainable by a method of the present invention.
  • Only pH manipulations and/or dilutions are performed as needed between the two chromatography steps in order to prepare the mixture for purification in the second step of chromatography.
  • the AEC step employs a membrane which uses a quaternary ammonium ion ligand and the CEC step employs a sulfonate-based ligand attached to a resin.
  • a low pH hold can be incorporated into the present method, as an in-process step, if desirable. Due to viral clearance requirements of regulatory agencies for the production of therapeutic proteins, there must be at least two separate steps for viral inactivation and clearance, based on different chemical modes of action which are typically a low pH hold and a viral filtration step.
  • the present invention can accommodate a low pH hold at any point, and before or after the AEC step. It shall be appreciated by those of ordinary skill in the art that a pH hold between the two chromatography steps can be accomplished using any methodology known in the art to accomplish the viral clearance typically achieved by a low pH hold, provided that the mixture containing the protein of interest is buffered appropriately prior to application of the AEC step.
  • the low pH hold typically results in an increase in conductivity of at least 0.5 mS/cm.
  • the resulting pH after the pH hold and neutralization is likely to be within a suitable pH range, which is about 4 to about 10, preferably about 6 to about 9. It shall be appreciated by those having ordinary skill in the art that a low pH hold step is not essential to achieve levels of purity attainable by a method of the present invention.
  • a low pH hold can be performed using, e.g, IN HCl, phosphoric acid or glacial acetic acid to decrease the CEC eluate to a pH range of about 3.4 to 3.6 followed by addition of, e.g., 2M Tris pH 9.0 to increase the eluate pH to about 7.0. It shall be appreciated by those of skill in the art that various acids and bases can be applied.
  • a low pH hold step used in a method of the present invention.
  • a low pH hold is performed for at least about 15 minutes, preferably about 30 minutes, more preferably about 45 minutes, still more preferably up to about 60 minutes, and most preferably up to about 90 minutes.
  • viruses can be inactivated by the addition of detergents and allowing contact time in solution according to conventional methods.
  • a second viral clearance step can be employed, e.g., a virus filtration step, which is, however, not required to achieve the levels of purity attainable by a method of the invention.
  • Filtration devices useful in viral clearance are well-known in the art (e.g., Ultipor® VF Grade DV20 or DV50 and Filtron® TFF (Pall Corporation, East Hills, N.Y.); Viresolve (Millipore, Billerica, Mass.); VR CUNO (CUNO; Meriden, Conn.); and Planova® (Asahi Kasei Pharma, Planova Division, Buffalo Grove, Ill.).
  • Viral filtration can be included at any point in the process, but is it typically performed once the product has been purified and the processing volume has been minimized.
  • Chromatography may be carried out in a column.
  • the column may be run with or without pressure and from top to bottom or bottom to top. If desirable, the direction of the flow of fluid in the column may be reversed during a chromatography process according to routine methods well known in the art.
  • Chromatography may also be carried out using a batch process in which the solid support is separated from the liquid used to load, wash, and elute the sample by any suitable means, including gravity, centrifugation, or filtration.
  • Chromatography may also be carried out by contacting the sample with a charged filter that absorbs or retains some molecules in the sample more strongly than others, using the same chemical principles as those described for a chromatography resin.
  • the sanitization step typically there is a hold, e.g., for about 15 to 30 minutes, preferably for about 1 hour, after the sanitization solution is applied onto the column in order to clean the resin having any bound contaminants, including contaminants of biological origin.
  • the charge step neutralizes the resin by displacing the sanitization solution, e.g., in CEC, NaCl can be used to displace NaOH from the column and maintain the resin ligand in contact with positively charged ions.
  • an equilibration buffer is used to equilibrate the column in order to prepare the pH and conductivity of the resin to bind the protein of interest.
  • the column can be considered equilibrated when its pH and conductivity are within ⁇ 0.1 and within ⁇ 0.2 mS/cm of the pH and conductivity of the equilibrating buffer, respectively.
  • a load mixture is prepared, which is typically a cell culture supernatant that is concentrated and buffer exchanged in an appropriate buffered salt (i.e. load buffer).
  • the load mixture i.e., containing the protein of interest for which purification is desired
  • the load mixture derived from the recombinant host cells is loaded onto the equilibrated column (CEC) using a loading buffer which optionally can be the same as the equilibration buffer.
  • the protein of interest and other impurities e.g., HCPs if the protein is produced in a CHO cell
  • bind differentially to the . solid phase thereby permitting separation as the protein and contaminants pass through the chromatography column.
  • wash steps using a wash buffer as described are performed to clear contaminants from the column.
  • a wash step can be performed at a slower flow rate than for the other wash and elution steps (but is not necessary), e.g., using a flow rate corresponding to several minutes (e.g., 2 to 30 minutes) residence time. Flow rates are discussed in greater detail infra.
  • the pH and conductivity of the wash buffer are important in order to maximize removal of HCPs.
  • the wash step in CEC removes nucleic acids and remaining HCPs while retaining the protein of interest.
  • an appropriate elution buffer is used, as described above, to cause the protein of interest to reverse binding off the column.
  • the type and concentration of buffer, salt, and/or other compound in the buffer composition are such that the protein of interest elutes differentially in relation to the impurity(ies).
  • the appropriate pH and conductivity ranges for loading, wash, and elution buffers can be readily determined by those having ordinary skill in the art, using the Example provided herein as guidance, such that the protein of interest is recovered during elution.
  • a reduced flow rate described infra.
  • the protein of interest As the protein of interest is removed from the column, it is collected based on formation of a peak from rising A 280 to falling A 280 .
  • the baseline can be readily determined by one of ordinary skill in the art by measuring the absorbance of the equilibrating buffer at 250-280 nm while the equilibrating buffer passes through the column.
  • eluate containing the protein of interest is collected before 25% of the maximum peak height.
  • the separation methods used in the present invention cause contaminants to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, while the protein of interest adheres to the chromatography medium, and is then differentially eluted depending on the buffer.
  • the reduced flow rate at which a mixture moves down the column is from about 2 to 10 minutes residence time, preferably no more than 30 minutes residence time.
  • the flow rate in the chromatography step corresponds to a residence time not greater than about 6 minutes. While the flow rate is not essential to achieving purity levels obtainable by a method of the invention, rates are provided for purposes of guidance. One having ordinary skill in the art can modify the flow rate as needed.
  • the preferred measure of protein purification efficiency by a process of the present invention is the measure of host cell protein removal.
  • the purified protein is preferably a homogenous composition as it is defined supra. Protein concentration of a sample at any stage of purification can be determined by any suitable method. Such methods are well known in the art and include: 1) colorimetric methods such as the Lowry assay, the Bradford assay, the Smith assay, and the colloidal gold assay; 2) methods utilizing the UV absorption properties of proteins; and 3) visual estimation based on stained protein bands on gels relying on comparison with protein standards of known quantity on the same gel. See e.g. Stoschek (1990), Quantitation of Protein, in Guide to Protein Purification, Methods in Enzymol. 182: 50-68.
  • Protein contamination of a homogenous composition containing the protein of interest can be determined by various means know in the art, e.g., by enzyme-linked immunosorbent assay (ELISA; see e.g. Reen (1994), Enzyme-Linked Immunosorbent Assay (ELISA), in Basic Protein and Peptide Protocols, Methods Mol. Biol. 32: 461-466, which is incorporated herein by reference in its entirety).
  • ELISA enzyme-linked immunosorbent assay
  • the amount of nucleic acid that may be present in a homogenous composition containing the protein of interest can be determined by any suitable method. For example, one can use an assay utilizing polymerase chain reaction. Nucleic acid can be reduced to levels less than 10pg/mg.
  • the protein thus recovered can be formulated in a biopharmaceutically acceptable composition according to routine formulation methods well known in the art and used for various diagnostic, therapeutic and other purposes requiring a highly purified protein composition.
  • any proteins that may remain bound to the column can be released by stripping the chromatography medium using a solution at a higher molarity and/or modified pH.
  • the column may then be regenerated using a solution (e.g., a regeneration solution) that will have the effect of releasing most or all proteins from the chromatography medium and reducing or eliminating any microbial contamination that may occur in the chromatography medium.
  • a solution e.g., a regeneration solution
  • the column may be rinsed and stored in a solution that discourages microbial growth.
  • a solution may comprise sodium hydroxide, but other reagents can also be used, such as ethanol, sodium azide, benzyl alcohol or high concentration of lyotropic salts.
  • the present Example describes the purification of antibodies using a purification method of the present invention (see FIG. 1 ).
  • Fully human antibodies were prepared against a tumor antigen gm.
  • Transfectomas were prepared using CHO DG44 cells. Cell culture was performed using synthetic, serum-free, defined medium at neutral pH (7 to 8) and conductivity between 10 and 20 mS/cm. The DG44 CHO cells were grown to a density of about 2 to 10 ⁇ 10 6 cells/ml. Cell culture was clarified by filtration using a 60MO2 CUNO filter (Meriden, CT). The resulting cell culture supernatant was concentrated and diafiltered in 70mM Sodium Phosphate, pH 6.2.
  • Tables 1 and 2 show a summary of the operational details of the two step purification process of the invention for production of therapeutic grade protein material.
  • the first non-affinity chromatography step was cationic exchange chromatography using a Fractogel EMD SE HiCap resin for capture, followed by a low pH hold using IN HCl or glacial acetic acid to decrease the pH of the eluate to 3.4 to 3.6 and 2M Tris pH 9.0 to increase the pH of the fraction to 7.0.
  • the process was carried out in the CEC step using equilibration, loading, wash and elution buffers for the respective resin as indicated in Table 1.
  • Column is equilibrated when pH and Wash 2 conductive are ⁇ 0.1 and ⁇ 0.2 mS/cm of the equilibrating buffer, respectively. Conductivity and pH ranges are important to bind the antibody, while minimizing the binding of host cell proteins.
  • the purification method did not use an in-process buffer exchange step, i.e., the sample proceeded from the first chromatography step to the second with only pH and dilution manipulations to perform the optional low pH hold.
  • the dilution, virus inactivation, virus removal and formulation steps were used in this Example for making a therapeutic grade protein composition. It shall be appreciated by those of skill in the art that none of these steps affects the purity of the protein product relative to other organic compounds.
  • the purification process of this Example achieved a homogenous composition containing less than 100 ppm host cell protein (see Table 3 below), less than 10pg DNA/mg antibody and greater than 99% monomer, which permits further processing and formulation of the purified material to a therapeutic grade product.
  • the contaminant concentration after the first step varied between 200 and 1500 ng/mg for CHOPs and 95 to 100% monomer. The results are reported in Table 3.

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WO2013022730A1 (fr) * 2011-08-05 2013-02-14 Veridex, Llc Nouveau procédés de couplage de molécules à des surfaces en métal/oxyde métallique
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US9505803B2 (en) 2009-12-18 2016-11-29 Novartis Ag Wash solution and method for affinity chromatography
US9719074B2 (en) 2012-06-29 2017-08-01 Shire Human Genetic Therapies, Inc. Cells for producing recombinant iduronate-2-sulfatase
US10344270B2 (en) 2012-06-29 2019-07-09 Shire Human Genetic Therapies, Inc. Methods and compositions for treatment of Hunter syndrome
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US20080226781A1 (en) * 2007-03-15 2008-09-18 Emsland-Starke Gmbh Method of Obtaining Vegetable Proteins and/or Peptides, Proteins Produced According to Said Method and/or Peptides and Use Thereof
US9505803B2 (en) 2009-12-18 2016-11-29 Novartis Ag Wash solution and method for affinity chromatography
WO2013022730A1 (fr) * 2011-08-05 2013-02-14 Veridex, Llc Nouveau procédés de couplage de molécules à des surfaces en métal/oxyde métallique
US11332514B2 (en) * 2011-08-25 2022-05-17 Hoffmann-La Roche Inc. Cation and anion exchange chromatography method
US9719074B2 (en) 2012-06-29 2017-08-01 Shire Human Genetic Therapies, Inc. Cells for producing recombinant iduronate-2-sulfatase
US10344270B2 (en) 2012-06-29 2019-07-09 Shire Human Genetic Therapies, Inc. Methods and compositions for treatment of Hunter syndrome
US11530393B2 (en) 2012-06-29 2022-12-20 Takeda Pharmaceutical Company Limited Compositions comprising iduronate-2-sulfatase
WO2015142777A1 (fr) * 2014-03-17 2015-09-24 Regeneron Pharmaceuticals, Inc. Clarification de culture cellulaire de mammifère
US20170191101A1 (en) * 2014-03-17 2017-07-06 Regeneron Pharmaceuticals, Inc. Clarification of mammalian cell culture
EP3119871A4 (fr) * 2014-03-17 2018-01-03 Regeneron Pharmaceuticals, Inc. Clarification de culture cellulaire de mammifère
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